Download Thin-Layer Chromatography: Applying TLC as a

Thin-Layer Chromatography:
Applying TLC as a Method to Monitor the Synthesis of Aspirin
Barbara Jennings
Chem 203.1
Desk 109.D
TA: Tian Chen
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Introduction:
Chromatography has been an effective means of separating a mixture of chemical
compounds for over a hundred years. Originally chromatography had been preformed by
separating components in a column of calcium carbonate. In 1938, Nikolai Izmailov and his
postgraduate student Maria Schreiber developed a newer form of chromatograph known today as
Thin-layer chromatography (TLC). TLC is a solid-liquid form of chromatography that consists
of a thin plastic backing coated with a polar solid absorbant, usually silica gel, and a mobile
phase consisting of organic solvents with varying polarities. TLC separates a mixture of two or
more compounds based on polarity. As the mobile phase solvent carries the compounds up the
plate through capillary action, the more polar compound will interact with the stationary phase
separating itself from the less polar compound that will stay in the mobile phase since it is less
attracted to the stationary phase. The mobile phase consists of a mixture of solvents that are just
polar enough to allow the polar compound to elute slightly, but just nonpolar enough to achieve a
greater separation by partition from the nonpolar compound (see figure 1, appendix A). This fast,
effective, and inexpensive means of chromatography has played a major role in food science,
medicine, and industry. One major application the pharmaceutical industries use TLC analysis
for is to monitor the synthesis of a drug or to identify the active ingredients of medicine.
Monitoring the reaction of a drug by TLC is very significant because it allows pharmaceutical
companies to know when the reaction is complete and also how pure their product is or if further
purification is needed due to the presence of impurities.
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TLC has been used to monitor the synthesis on many medicines, but one particular over
the counter drug has been aspirin. Aspirin, also known as acetylsalicylic acid, was invented in
1893 by Felix Hoffman originally as a treatment for arthritis pain and inflammation. Aspirin is
now commonly used as an anti-inflammatory, analgesic, and antipyretic. Acetylsalicylic acid is
synthesized by acetylating the phenolic functional group of salicylic acid with acetic anhydride.
This reaction is a similar mechanism to Fisher esterification or otherwise known generally as a
nucleophilic acyl substitution mechanism. The final product is formed protonating the acetic
anhydride, with the catalyis phosphoric acid, forming an electrophile that can then bond to the
nucleophilic phenolic substituent of salicylic acid. The attached acetic anhydride is then broken
apart leaving the acetyl group attached to the subsituent thus forming the final product
acetylsalicylic acid and byproduct acetic acid (see mechanism Figure 2, appendix B).
Purification of the final product can be achieved by recrystallization in a slightly polar solvent,
such as ethyl acetate. To determine if the synthesis has gone to completeness, the reaction
mixture is spotted on a TLC plate every 15 minutes and developed in a TLC chamber with a
mixture of ethyl acetate and hexane as the mobile phase until only one spot is observed in the
reaction mixture lane. Further analysis, by nuclear magnetic resonance (1H-NMR) spectroscopy,
is used to determine the purity of the final product.
The purpose of this experiment was to determine what mixture of ethyl acetate and
hexane will achieve the greatest separation of salicylic acid and acetylsalicylic aid. Then use this
solvent system to monitor the synthesis reaction of acetylsalicylic acid from the reactants
salicylic acid and acetic anhydride. Finally the purity of the final product, acetylsalicylic acid,
was determined by melting point analysis and by.
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Experimental:
TLC Mobile Phase Determination
An appropriate solvent system for TLC mobile phase to achieve a large separation of
salicylic acid and acetylsalicylic acid was determined by trial and error. Eight trials of hexane
and ethyl acetate were mixed in various percents (see Table 1). Each mixture was used as a
mobile phase to develop a TLC plate spotted with standards salicylic acid (1%) and
acetylsalicylic acid (1%) in a saturated TLC chamber. The mobile phase was allowed to develop
up the plate until the solvent line reached approximately one centimeter from the top of the TLC
plate. The plate was allowed to dry and then examined under ultraviolet (UV) light to visualize
the spots. The retardation factor (Rf) value was determined for each spot (see sample calculation
Figure 3, appendix A). The goal was to determine which solvent system created the best
separation of the standards. It was determined that trail four, 50% ethyl acetate and 50% hexane,
achieved the greatest separation of salicylic acid (Rf 0.945) and acetylsalicylic acid (Rf 0.291)
because it had the largest difference in Rf values (0.654).
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Mobile Phase
Mixture
Composition
Number
1
% Ethyl
Acetate
% Hexane
Rf
Salicylic
Acid
Rf
Acetylsalicylic
Acid
100
0
0.530
0.545
2
90
10
0.620
0.600
3
70
20
0.255
0.291
4
50
30
0.945
0.291
5
30
50
0.360
0.280
6
20
70
0.270
0.250
7
10
90
0.182
0.164
8
0
100
0
0
Table 1: Mobile Phase Mixture combinations for the separation of Salicylic Acid (1%) and
Acetylsalicylic acid (1%)
Synthesis of Aspirin
Synthesis of aspirin took place by placing salicylic acid (100mg, 0.724 mmol) in a 13 x
100 mm test tube with acetic anhydride (0.25 mL, 2.25mmol) and one pipet drop of phosphoric
acid (85%). The tube was corked, gently mixed and placed in a warm beaker of hot water just
below boiling point. After every 15 minutes the reaction was checked by TLC using a mobile
phase 50/50 mixture of ethyl acetate/hexane and a TLC plate spotted with three lanes: pure
salicylic acid (1%), pure acetylsalicylic acid (1%), and the reaction mixture (R). Rf values were
calculated for each spot that formed. The reaction is said to have synthesized to completeness
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when there is only one spot left, the acetylsalicylic acid spot, meaning there is no more salicylic
acid present.
Figure 4: TLC plates used to monitor the synthesis if acetylsalicylic
acid at 15, 30, and 45 minutes. Lane S: Salicylic Acid (1%)
Standard. Lane R: Reaction Mixture. Lane AS: Acetylsalicylic Acid
(1%) Standard
Time (minutes)
Salicylic
acid Rf
Acetylsalicylic
acid Rf
0.346
Reaction
Mixture Rf
Lower Spot
0.385
Reaction
Mixture Rf
Upper Spot
0.481
15
0.385
30
0.320
0.340
0.360
0.500
45
0.264
0.321
0.321
0.491
Table 2: Retardation factor (Rf) values for TLC of salicylic acid, acetylsalicylic acid, and
reaction mixture in a 50/50 ethyl acetate/hexane solvent system over time.
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After 45 minutes the tube was removed for the warm water bath and distilled water (0.7
mL) was added to quench the reaction. Slow cooling allowed crystals to form and vacuum
filtration removed the reaming solvent. The crystals were allowed to dry for one week and
melting point was then taken three times to determine purity of crystals. Further purification was
not need since. Acetylsalicylic acid was obtained as flaky white crystals (97mg, 75% yield); mp
135-136° C (lit. acetylsalicylic acid 134-136° C, lit. salicylic acid 158-161° C) and a 1H-NMR
spectrum was obtained ( see Figure 5 (Appendix C) and Table 3) .
Shift (ppm)
Splitting Observed
Integral Observed
11.649 ppm
7.074-8.206 ppm
2.346 ppm
Singlet
Multiplet
Singlet
1.00
4.35
3.00
Type of hydrogen
-COOH
Ar-H
H3-C-COOR
Table 3: 1H-NMR data, obtained for Reaction sample (35mg) with deuterated chloroform solvent on a
60 MHz 1H-NMR.
Sample calculation for percent yield of acetylsalicylic acid:
%yield = (actual yield / theoretical yield) x 100
75% = (97mg / 130mg) x 100
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Results, Discussion, Conclusion:
When determining the best mobile phase to monitor the synthesis of aspirin, it was
important to find the correct combination of ethyl acetate and hexane that would produce the
highest separation of salicylic acid and acetylsalicylic acid. However, as seen in Table 1, many
different results occur depending on the ration of ethyl acetate to hexane, only the right
combination can achieve significant separation between the reactants and products for the
synthesis of aspirin. For example, when using pure hexane the entire solvent has a nonpolar
composition. Therefore acetylsalicylic acid, the more nonpolar compound, will be more attracted
to the mobile phase and move with the hexane solvent while salicylic acid, the more polar
compound, will be extremely attracted to the polar stationary phase and will not move at all
(Table 1). When using a solvent system that is %100 ethyl acetate both the acetylsalicylic acid
compounds and salicylic acid elute too fast since the nonpolar compounds are not attracted to
either the stationary phase or mobile phase, while salicylic acid will elute just as fast since the
compounds are more attracted to the mobile phase than the stationary phase. From the results, a
50/50 mixture of ethyl acetate and hexane achieve the greatest theoretical separation of
acetylsalicylic acid and salicylic acid because there is a greater distance between the developed
spots and has the biggest difference in Rf values (0.654).
Using solvent system should theoretically produce the greatest separation of the products
and reactants when monitoring the synthesis of aspirin. However, this mobile system did not
produce ideal separation of products and reactants. The salicylic acid spot of the reaction mixture
was expected to have a much lower Rf value than the acetylsalicylic acid, since the salicylic acid
appears to have a much higher polarity for the stationary phase due to the phenolic substituent.
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However, salicylic acid and acetylsalicylic acid were only separated slightly from one another,
having an Rf difference of 0.096, much lower than the theoretical separation of the standards (Rf:
0.654) (Table 2). One possible explanation for this occurrence may be due to internal physical
interactions of the salicylic molecules with each other in the reaction mixture. This may be
because the hydrogen on the phenolic substituent of salicylic acid interacts with the double
bonded oxygen of the other substituent, carboxylic acid, through hydrogen bonding thus
decreasing its polarity significantly. Due to higher interaction among molecules, salicylic acid is
less attracted to the stationary phase and will move further up the plate thus having a higher Rf
value than the standard salicylic acid and a closer Rf value to the acetylsalicylic acid in the
reaction mixture. Another complication that occurred was that the reaction mixture had an upper
spot that consistently higher than both the standards it was develop with (Figure 5). This may be
due to the fact that acetylsalicylic acid in the reaction mixture was had many other molecules
interacting and blocking the acetylsalicylic acid. Thus it’s polar carboxylic acid substituent had
less chance to interact with the stationary phase than the pure standard acetylsalicylic acid.
As time progressed the TLC plates showed no sign that the reaction had run to
completeness. There were still two separate spots that appeared on the TLC plate after 45
minutes. This may be due to the fact that even though the reaction may have been completed, the
acetylsalicylic acid compounds were actually more polar than expected, due to the carboxylic
acid substituent, causing a greater attraction to the polar stationary phase and streaking in two
close spots. Since the Rf value of the lower spot (0.321) had the exact same Rf value of the
standard acetylsalicylic acid (0.321), this confirmed that the final TLC plate had only
acetylsalicylic acid was left in the reaction mixture, thus the reaction was complete. Since the
melting point (134-135° C) was very close to the actual melting point of acetylsalicylic acid
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(134-136° C) there was no further purification needed. Isolating the final product, acetylsalicylic
acid, was a success. Further 1H-NMR spectrometry analysis was preformed to confirm the
identity of the product.
The 1H-NMR spectrum data matched perfectly to the theoretical peaks that would
be seen if pure acetylsalicylic acid 1H-NMR data were obtained (Figure 6, Appendix D). There
was a small peak at 11.649 ppm with an integral value of 1.00 due to the carboxylic acid
hydrogen, multiple peaks ranging form 7.074-8.206 ppm with an integral value of 4.35
indicating the four hydrogens on the aromatic ring, and a significant single peak at 2.346 ppm
with an integral value of three indicating the hydrogens on the ester (-COOCH3) substituent. The
peak at 0.00 ppm is a TMS peak, typical when using the solvent deuterated chloroform. There
were no other significant peaks presented in the spectrum, indicating the isolated product was
pure. The obtained spectrum is a near perfect match with the theoretical spectrum of standard
acetylsalicylic acid. For these reasons it is reasonable to confirm that an esterification reaction
that took place between salicylic acid and acetic anhydride formed pure acetylsalicylic acid. The
percent yield supports that fact that the synthesis of aspirin was successful because the product
obtained had a high yield of 97 mg (75% yield) from a theoretical yield of 130mg. Some residual
crystals may have been left on glassware, accounting for the decrease in yield.
All in all the 50/50 mixture of ethyl acetate and hexane did not prove to be helpful in
monitoring the progression of the synthesis of aspirin. Since the spots were visualized as streaky
and close together is was difficult to tell whether the reaction was complete or there were still
reactants present. For future experimentation, more trial and error solvent systems should be
tested to determine the best solvent system that will stop internal physical interactions while
provide clear separation of components on the TLC plate. Melting point and 1H-NMR analysis
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were very helpful when determining the true identity of the product. The synthesis of aspirin
proved to be successful at achieving a high yield final acetylsalicylic acid product. In
conclusion, the techniques, knowledge, and skills gain from this experience have not only proven
to be helpful for this experiment, but also for future scientific work.
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References:
1. Bortiatynski, J.; Masters, K.; Minard, B.; Halmi, T.O.; Williamson, K.L. Lab Guide for
Chemistry 203 & 213B, 2009-2010, pp. 165-188.
2. Kapp, Khail. Chapter 7, Thin Layer Chromatography, March 2, 2010.
3. Kaine, Mary Ann. Chapter 7, Thin Layer Chromatography, March 2, 2010.
4. McMurray, J.; Simanek, E. Fundamentals of Organic Chemistry, 6th ed.; Thomson
Brooks/Cole: Belmont, CA, 2007, pp. 307-308.
5. Sigma-Aldrich. Acetylsalicylic Acid. http://www.sigmaaldrich.com/united-states.html.
(accessed March 28, 2009).
6. Young, Wei. Chapter 7, Thin Layer Chromatography, March 2, 2010.
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Appendix A:
Figure 1: Sample thin-layer chromatography (TLC) plate,
sample C shows the compound with the least polarity;
sample A shows the compounds with the most polarity
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Appendix B
Figure 2: Nucleophilic acyl substitution mechanism for the synthesis of
acetylsalicylic acid from salicylic acid and acetic anhydride
Retardation Factor (Rf) = distance spot travels
/
distance solvent travels
Example: Table 1, Mobile Phase Mixture Composition Number 1, salicylic acid
Rf = 2.9cm/5.5cm
= 0.53
Figure 3: Retardation Factor (Rf) theoretical and sample calculation.
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Appendix C
TMS
Figure 5: Annotated 1H-NMR spectral graph for the product of synthesized of acetylsalicylic
acid
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Appendix D
Figure 6: Predicted chemical shifts, splitting patterns, and integral values of pure salicylic acid
and acetylsalicylic acid.
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